Tacrolimus, also known as FK506, is the generic name for a macrolide immunosuppressant produced by the bacterium Streptomyces tsukabaensis, in the soil (See, Inamura, N., et al., Transplantation, 45(1):206-209 (1988)). The first generation major metabolites of tacrolimus are 13-O-demethylated tacrolimus (“M-I”), 31-O-demethylated tacrolimus (“M-II”), and 15-O-demethylated tacrolimus (“M-III”). Tacrolimus has been used intravenously and orally for the prevention of organ rejection, particularly in patients receiving liver, kidney or bone marrow transplantation.
Cyclosporine (“CsA”) is an immunosuppressive drug obtained from certain soil fungi. While primarily used to prevent organ rejection after transplant, CsA also has been used to treat other illnesses, such as aplastic anemia, or to prevent graft versus host disease (GVHD).
Tacrolimus has an in vivo potency 50-100 times greater than cyclosporine CsA (See, Murthy, J. N., et al., Clinical Biochemistry, 31(8):613-617 (1998)). The immunosuppressive effect of tacrolimus is similar to CsA and is thought to be through the selective inhibition of the generation of cytotoxic T cells. Id. At the molecular level, tacrolimus appears to selectively block the early transcriptional activities in the T-cell response. Id. This action of tacrolimus is attributed to the binding of drug to specific cytosolic proteins called immunophilins to form a complex. Id. This complex interacts with calcium dependent calcineurin-calmodulin translocation pathways and inhibits the nuclear translocation of a transcriptional factor (“NF-AT”), which binds to an enhancer polynucleotide sequence of the IL-2 genes needed for the transcription of IL-2 mRNA.
Clinically, tacrolimus is known to reduce rejection episodes in transplant patients. Although therapeutically beneficial, tacrolimus exhibits some toxicity similar to that of CsA, which includes nephrotoxicity, gastrointestinal tract complications and neurotoxicity. Id. Unlike CsA, tacrolimus does not cause hirsutism or hypercholesterolemia. Id. In view of the toxicity issues related to tacrolimus, immunoassays are used to monitor the blood concentrations of tacrolimus in patients receiving treatment with this drug.
A variety of different diagnostic immunoassays are commercially available for monitoring the blood concentrations of tacrolimus. Several of these immunoassays use organic solvents to extract the tacrolimus from whole blood samples. The organic solvent increases the equilibrium dissociation constant (KD) and/or lowers the functional activity of the antibody used in the assays. The reduced activity of the antibody leads to lower assay sensitivity and potentially lowers accuracy and robustness. Attempts have been made to increase assay sensitivity by reducing the amount of organic solvent used during the extraction process. However, reducing the amount of the solvent was found to impact the extraction efficiency and hence the assay reproducibility.
Likewise, a variety of different diagnostic immunoassays are commercially available for monitoring the blood concentrations of CsA, e.g., utilizing an anti-cyclosporine antibody. Current literature suggests that the generation of CsA metabolites can mask the concentration of active parent drug (CsA). The appropriate dosage of CsA immunosuppressant is critical for organ transplantation patients.
Therefore, there is a need in the art for new antibodies that have improved binding characteristics (such as affinity and specificity) that can be used in such diagnostic immunoassays. There also is a need for methods of screening for and obtaining such antibodies.